Expression control polynucleotides derived from spliceosomal protein gene promoters

ABSTRACT

An expression control polynucleotide capable of affecting the expression of a second polynucleotide, and derived from a spliceosomal protein gene promoter. Isolation of plant spliceosomal protein gene promoters from potato and maize is described. Partial sequences of the promoters of two potato spliceosomal protein gene promoters are disclosed.

This invention relates to expression control polynucleotides derivedfrom spliceosomal protein gene promoters.

It is already well known that organisms such as plants or animals withnovel characteristics can be produced by introducing genes or DNAsequences from the same or a related organism or by introducing genes orDNA sequences from other organisms. The key to production of novelphenotypes is the active expression of at least a part of the introducedDNA sequence. Generally expression of the introduced DNA sequence willoccur only in the presence of an expression control polynucleotide, suchas a promoter, which is compatible with the host organism. Promoters arenucleic acid sequences which are currently believed to regulate theexpression of a gene by facilitating the binding of proteins requiredfor transcription, such as RNA polymerase, to a portion of the nucleicacid sequence upstream of the gene. Thus for protein coding genes theDNA is transcribed into mRNA (messenger RNA) which is then relocated tothe cytoplasm where it is available for translation into polypeptide.

Whereas promoters are generally disposed upstream of the genes theyregulate and are thought to act by providing a binding site for an RNApolymerase transcription complex, another form of expression controlpolynucleotide known as an "enhancer" sequence can control theexpression of a gene usually without regard to its relative position ororientation.

Promoters can vary in levels of expression induction and in theirexpression patterns. Some promoters are active in a tissue-specific ordevelopmental stage-specific manner while other promoters are activeconstantly to continually drive expression of the gene they control.Such constantly active promoters are called constitutive promoters. Thepromoters of this latter class most widely used in genetic engineeringare the Cauliflower Mosaic Virus (CaMV) 35S RNA promoter, nopalinesynthetase (nos) or octopine synthetase (ocs) promoters. Constitutivepromoters induce gene expression at a relatively constant rate.

Although the above conventional promoters generally drive expression athigh levels, many have the disadvantage that they are derived from plantinfectious agents; the CaMV 35S RNA promoter is derived from a plantvirus and the other promoters mentioned are derived from strains ofAgrobacterlium, soil-borne infectious bacteria. The source of thesepromoters is a cause of concern in transgenic plant production. Inaddition, detailed analysis of expression patterns of CaMV 35S promoter,have shown that its levels of expression can vary greatly amongdifferent plant tissues to the level where it is inactive in sometissues and is therefore, no longer constitutive for such tissue. Formany biotechnological objectives, constitutive expression in all cellsand tissues would be of great advantage.

Splicesomal proteins are believed to be present in virtually alleukaryotic cells and are involved in the phenomenon of pre-mRNA splicingwhich removes introns (non-coding regions) from RNA transcripts beforeprotein production.

The present invention seeks to provide a promoter (or other expressioncontrol polynucleotide) which is not derived from an infectious agentand which is suitable for use in the control of expression ofrecombinant genes in the construction of transgenic organisms such asplants and animals.

The present invention also seeks to provide a promoter (or otherexpression control polynucleotide) which is likely to be activethroughout all or most cells of the organism.

According to the present invention there is provided an expressioncontrol polynucleotide at least partially derived from a spliceosomalprotein gene promoter.

The expression control polynucleotide of the invention is capable ofcontrolling the expression of a second polynucleotide (preferablycomprising a polypeptide-encoding sequence) operably linked thereto.

RNA sequences which do not code for protein can also be expressed egribozymes or anti-sense RNA.

The term "expression control polynucleotide" as used herein will includepromoters, enhancers or any other functional equivalents or any othersequence elements which affect expression of other gene sequences.

The term "polypeptide-encoding sequence" as applied herein topolynucleotides means a polynucleotide comprising a sequence which canbe transcribed into mRNA, which itself can be translated into apolypeptide. The "polypeptide-encoding sequence" may includenon-translated portions, such as introns.

The spliceosomal protein gene promoter may be derived from plants. Theplants may be dicotyledonous (eg potatoes), or monocotyledonous (egmaize).

The present invention also provides a recombinant polynucleotidecomprising an expression control polynucleotide according to theinvention operably linked to a second polynucleotide (preferablycomprising a polypeptide-encoding sequence).

The present invention also provides a recombinant vector containing anexpression control polynucleotide or a recombinant polynucleotide asdefined above.

According to the present invention there is also provided a method ofproducing a recombinant vector, said method comprising ligating anexpression control polynucleotide into a vector or part thereof. Amethod of producing a transformed cell by transfecting a host cell usingsaid recombinant vector forms another aspect of the invention.

The present invention also provides a transformed host cell containing arecombinant polynucleotide or vector as defined above.

The present invention also provides a transgenic organism (for example atransgenic plant) containing a recombinant polynucleotide or vector asdefined above. The progeny (and seeds) of such transgenic organismsforms a further part of the invention.

The present invention also provides a method for controlling theexpression of a polypeptide from a nucleotide sequence encoding thepolypeptide, said method comprising operably linking said sequence to anexpression control polynucleotide of the invention.

The expression control polynucleotide of the invention may comprisedouble- or single-stranded DNA or RNA.

Three cultures of E.coli (SCRI/JB/1, SCRI/JB/2 and SCRI/JB/3), eachcontaining a plasmid having an expression control polynucleotideaccording to the invention were deposited on Mar. 14, 1994 with theNational Collection of Type Cultures under numbers NCTC 12864, NCTC12865 and NCTC 12866 respectively.

Cultures SCRI/JB/1 and 2 contain dicotyledonous spliceosomal proteingene expression control polynucleotides (promoters for potato U1A andU2B" genes respectively); SCRI/JB/3 contains a monocotyledonousexpression control polynucleotide (promoter for a maize PRP8 gene).

Accordingly, the present invention also provides NCTC deposits Nos12864, 12865 and 12866 and the plasmids thereof.

Recombinant DNA technology has been recognised as a powerful techniquenot only in research but also for commercial purposes. Thus, by usingrecombinant DNA techniques (see Sambrook et al 1982 and "Principles ofGenetic Engineering", old and Primrose, 5th edition, 1994) exogenousgenetic material can be transferred to a host cell and the polypeptideencoded by the exogenous genetic material may be replicated by and/orexpressed within the host. For the purposes of simplicity recombinantDNA technology is normally carried out with prokaryotic micro-organisms,for example bacteria such as E. coli, as host. However, use has alsobeen made of eukaryotic organisms, in particular yeasts or algae, and incertain applications eukaryotic cell cultures may also be used.

Genetic alterations to mammalian species by micro-injection of genesinto the pro-nuclei of single-cell embryos is also well known and hasbeen described by Brinster et al, in Cell 27: 223-231, 1981. Thusgeneral techniques used in recombinant DNA technology and the productionof transgenic organisms is within the scope of the skilled man.

Using a cDNA sequence (complimentary DNA--reverse transcribed frommRNA), either full-length or partial, as a probe for a gene of interest,the gene promoter can be readily isolated by standard procedures.Briefly, the cDNA probe is first used to screen a genomic library toisolate a genomic clone containing the promoter and coding sequence.Restriction mapping and Southern blotting with the cDNA as probedelineates the region of the genomic clone containing the codingsequence. Sequencing of this region of the genomic clone and comparisonto the cDNA clone will identify the translation initiation ATG codon,and if the cDNA is full-length, will give an indication of thetranscription start site, upstream of which lies the gene promoter.Important promoter sequence elements may lie in excess of around 2 kbpof the transcription start site. Therefore, a genomic fragment ofpreferably 1-5 kbp is isolated for initial testing of promoter activity.For example, in the case of U1A and U2B", a monoclonal antibody (mAb),4G3, raised against a b-galactosidase-human U2B" protein, was used as aprobe to screen a potato cDNA expression library and a full-length cDNAclone was isolated (Simpson et al. 1991). Screening of a potato genomiclibrary using this cDNA clone resulted in a genomic clone containingpart of the gene and around 15 kbp upstream sequences which were used toclone the promoter in subsequent experiments. The extreme similaritybetween U1A and U2B" enabled us to isolate a full-length genomic cloneof the U1A gene. Since the latter clone contained around 7 kbp ofupstream sequences, it was possible to clone the promoter from theupstream region.

The use of spliceosomal protein gene promoters to drive expression ofDNA sequences in transgenic plants or animals has the advantage that theexpression is constitutive, expressed in all cell and tissue types anduses naturally occurring plant or animal nucleic acid sequences whichare not derived from infectious agents.

While further modifications and improvements may be made withoutdeparting from the scope of this invention, the following is adescription of one or more examples of the invention, with reference tothe accompanying drawings, in which:

FIG. 1 shows a schematic diagram of a potato U2B" genomic clone presentin the cells of culture No SCRI/JB/2;

FIG. 2 shows a schematic diagram of a potato U1A genomic clone presentin the cells of culture No SCRI/JB/1;

FIG. 3 shows a schematic diagram of a maize PRP8 genomic clone presentin the cells of culture No SCRI/JB/3;

FIG. 4 shows a schematic diagram of a UIA/GUS expression cassette;

FIG. 5 shows a schematic diagram of a U2B"/GUS expression cassette;

FIG. 6 shows a graph of fluorescence against time plotted from datacollected from a fluorometric assessment of GUS gene expressioncontrolled by potato spliceosomal protein gene promoters;

FIG. 7 shows a schematic diagram of a UIA/GUS expression cassette;

FIG. 8 shows a schematic diagram of U2B"/GUS expression cassette;

FIG. 9 shows a schematic diagram of the genomic organisation of thepotato U1A gene; and

FIG. 10 shows a schematic diagram of the genomic organisation of thepotato U2B" gene.

EXAMPLE 1

Genes for two plant spliceosomal proteins from the dicotyledonous plant,potato (Solanum tuberosum) were isolated encoding the spliceosomalproteins U1A and U2B".

The promoter of a potato U2B" gene was isolated from a potato genomiclibrary in λ EMBL 3 by conventional methods. The library was screenedwith a potato U2B" cDNA clone (Simpson et al., 1991) using standardprocedures (Sambrook et al., 1989). The potato U2B" genomic clone wasplaque-purified, DNA prepared, fragments subcloned into plasmid vectorsand DNA sequenced by standard procedures (Sambrook et al. 1989).

The genomic clone contained an insert of 15 kilobase pairs (kbp)(FIG. 1) from which relevant sub-fragments were cloned into plasmidvectors (FIG. 1). The clone only contained a fragment of coding regionof U2B" (100 bp), a fragment of the first intron in U2B" (100 bp) andapproximately 15 kbp of upstream sequences containing the U2B" promoter.

EXAMPLE 2

The promoter of a potato U1A gene was isolated from a potato genomiclibrary in λ EMBL 4. The library was screened with a potato U2B" cDNAclone (Simpson et al., 1991) because U1A and U2B" are closely related,and a genomic clone was obtained by known methods. The genomic clone wascharacterised by state-of-the-art methodologies as described by Sambrooket al., (1989). The clone contained an insert of 15 kbp (FIG. 2) fromwhich relevant sub-fragments were cloned into plasmid vectors bystandard techniques (Sambrook et al., 1989). The clone contained thewhole of the U1A coding sequence on five exons, four introns and 7 and 2kbp of 5' and 3' flanking sequence respectively.

The promoter regions can be linked to marker genes such as bacterialβ-glucuronidase (Jefferson, 1987) by standard molecular techniques(Sambrook et al., 1989). Promoter constructs can be analyzed byintroduction into plant cells by known methodology such as chemical orelectrical transfection, microinjection, biolistics orAgrobacterium-mediated or other vector-mediated transformation (seeShaw, 1988). Transgenic plants containing the construct can be analyzedby detecting the presence of GUS enzyme and thereby its expression usingknown methods of histochemical staining (Jefferson, 1987). Levels ofexpression of marker genes driven by the promoters in either stablytransformed plants or transiently transformed plant cells or protoplastscan be assessed by comparison with levels of endogenous gene expressionand of marker gene expression driven by the Cauliflower Mosaic Virus 35SRNA promoter. This analysis can use known, state-of-the-artmethodologies to detect RNA transcripts (Sambrook et al., 1989; Simpsonet al., 1992) and to detect production of enzyme from marker genes, forexample, for GUS marker gene activity.

The promoter region can be incorporated into plasmid vectors designedfor general use in construct production in E.coli, and for use instable, Agrobacterium-mediated transformation and in transienttransformation or stable, physical transformation methods. DNA sequencesto be expressed in the transgenic plant can be inserted behind thepromoter regions as is currently commonly performed using the CaMV 35SRNA promoter (see Shaw, 1988) prior to introduction into plant cells orproduction of transgenic plants.

EXAMPLE 3

A gene for a plant spliceosomal protein was cloned from themonocotyledonous plant, maize (Zea mays L.), which encodes thespliceosomal protein PRPS (Jackson et al., 1988) by virtue of sequencehomology to PRP8 of yeast.

The genomic clone of maize PRP8 was isolated from a maize genomiclibrary constructed in λ EMBL 4 by conventional methods. The library wasscreened with a fragment of maize PRP8 generated by state-of-the-artmethodologies, such as polymerase chain reaction (PCR) amplificationusing oligonucleotide sequences designed from the yeast andCaenorhabditis elegans PRP8 DNA sequences, cloning and sequencing. Themaize PRP8 genomic clone was plaque purified, DNA prepared and fragmentssubcloned into plasmid vectors by standard procedures.

The PRP8 promoter region can be incorporated into plasmid vectors aspreviously described.

EXAMPLE 4

Promoter/GUS Constructions

Two expression cassettes containing the β-glucuronidase (GUS) genedriven by the potato U1A and U2B" splicesomal protein gene promotersrespectively were constructed. Both cassettes contained the nopalinesynthase poly A (NOS-ter) downstream to the GUS gene. The constructionof these expression cassettes was achieved by replacing the CaMV 35Spromoter in the vector pBI221 by upstream sequences of the potato U1Aand U2B" spliceosomal protein genes. Genomic clones described above wereused.

UIA

A 4.5 kb PstI fragment containing about 2.5 kb upstream to the U1A gene,the first and the second exons, the first intron and part of the secondintron was used to clone the U1A promoter. In order to clone thepromoter, a site specific mutation was generated which changed thesequence ATGGCG at the translation start site into TCTAGA to introducean XbaI restriction site. Subsequently, the entire 2.5 kb upstreamregion was cloned into pBI221 after eliminating the 35S promoter usingthe restriction enzymes PstI and XbaI (FIG. 4).

U2B"

An EcoRI/SalI fragment containing about 2 kb upstream to the U2B" gene,the entire first exon, and part of the first intron was used to clonethe U2B" promoter. Appropriate restriction sites were introduced intothe upstream region using PCR amplification. The first PCR primerstarted a few nucleotides downstream of the EcoRI site and containedadditional nucleotides in its 5' end to provide PstI and BglII sites.The second primer started two nucleotides upstream of the ATG startcodon and contained additional nucleotides providing a site for cleavageby BamHI at its 5' end. After PCR amplification and digestion with BamHIand PstI, the 2 kb upstream region was cloned into pBI221 replacing the35S promoter (FIG. 5).

Protoplast Isolation

The constructed cassettes were tested for promoter activity in transientgene expression assays using tobacco protoplasts. Protoplasts wereprepared from young, fully expanded tobacco leaves. Leaves were placedin 7% Domestos for 10 minutes, washed with sterile tap water, dried, andpeeled to remove the lower epidermis. Peeled leaf pieces were placedonto 15 ml enzyme solution in a sterile Petri dish [enzyme solution: 1mg/ml cellulase, 0.5 mg/ml driselase, 0.2 mg/ml macerase, suspended inTO⁻ (see appendix)] and incubated overnight in the dark at 25° C.Protoplasts were transferred into two sterile 10 ml tubes throughsterile sieves and spun at 400 rpm for 5 minutes. After resuspendingeach protoplast pellet in 10 ml TO⁻, 5 ml aliquots were each layeredonto 2.5 ml 16% sucrose and spun at 800 rpm for 5 minutes. The purifiedprotoplasts were resuspended in 10 ml TO⁻, spun at 400 rpm for 5minutes, and recollected in 5 ml TO⁻.

Protoplast Transfection

Transfection of the protoplasts with the U1A/GUS and U2B"/GUS constructswas achieved using plasmid DNA purified by Qiagen (Trade Mark) columns.For each experiment 200 μl of the purified protoplasts were transfectedwith 30 μg DNA dissolved in 20 μl distilled water. After dropwiseaddition of the DNA and careful homogenization of protoplasts, 200 ml ofPEG solution was added. [PEG solution: 25% PEG 8000, 0.1M Ca(NO₃)₂,0.45M Mannitol, 10 mM MES: 2-(N-Morpholino) ethanesulfonic acid] wasadded dropwise. The transfected protoplasts were then incubated atambient temperature for 20 minutes; subsequently, 4 ml calcium nitratewas carefully added [0.275 M Ca(NO₃)₂ ]. After an incubation period of20 minutes at ambient temperature protoplasts were spun at 400 rpm for 3minutes, suspended, and resuspended in 5 ml TO⁺. The transfectedprotoplasts were then incubated in sealed Petri dishes in the light at25° C. for 48 hours.

GUS Assays

Samples were collected by spinning the protoplasts at 400 rpm for 3minutes, resuspended, transferred into Eppendorf tubes (1 ml each), andspun again at 1000 rpm for 2 minutes in a cold microcentrifuge (4° C.).The pellet was then resuspended in 200 μl extraction and reaction buffer(50 mM NaPO₄ pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 10 mMβ-mercaptoethanol), spun at 1300 rpm for 3-5 minutes, and thesupernatant was stored in clean Eppendorf tubes. 50 μl sample extractwas added to 220 μl extraction and reaction buffer in an eppendorf tubeand preincubated at 37° C. for 5 minutes. At 15 sec intervals, 30 μl 1mM MUG (4-methyl umbelliferyl glucuronide) substrate was added to eachtube giving time to stop reactions at accurate intervals. At definedtime points, 100 μl samples were taken and added to 2.9 ml stop solution(0.2 M Na₂ CO₃) in a clean cuvette. At this stage, GUS activity could beinspected visually by transillumination using a long wave UV light box.Quantitative measurements of fluorescence were made using a fluorometer.Protoplast transfections and GUS assays have been performed toinvestigate the expression of the GUS gene driven by splicesomal proteingene promoters using the above described constructs. In all experiments,the vector pBI221 which contains the GUS gene driven by the CaMV 35Spromoter and terminated by the nopaline synthase poly A was used as apositive control. Negative controls were a: extracts from protoplaststransfected with water instead of plasmid DNA; and b: MUG substrateadded to the extraction and reaction buffer without the addition of anyextracts. Reactions were carried out for 30 minutes, 1 hour, 2 hours, 3hours, 4 hours, 5 hours and 20 hours. In order to assess the activity ofthe tested promoters in comparison to the 35S promoter, fluorometerreadings were set at 100 for pBI221 at each time point so that thereadings obtained for U1A and U2B" indicated the GUS activity in eachcase relative to that obtained for the 35S promoter. The results whichare illustrated in FIG. 6 show that the U2B" promoter has about 70% ofthe activity of the 35S promoter whereas the U1A promoter gave rise to10-15% of the activity observed for the 35S promoter.

FIGS. 9 and 10 show the genomic organisation of the original genomicclones made of potato genes U1A and U2B". The promoter of the U1A geneis believed to lie in the region between the ATG initiation codon andthe PstI site 2.5 kb upstream of the coding sequence initiating at theATG start codon. The promoter of the U2B" gene is believed to lie in theregion between the ATG coding region and the EcoRI site 2.0 kb upstreamof the coding sequence initiated by the ATG start codon.

FIGS. 9 and 10 indicate regions of the genomic clones containing thepromoters of U1A and U2B" which have been sequenced. The sequences ofthose regions are shown in the sequence listing.

The partial U1A promoter sequence is shown on the sequence listing asSEQ:ID:No1. Sequence data is presented for the 3' end of the regionbetween the ATG codon and a Pst1 site 2.5 kb upstream. About 1.7 kb ofthe 5' end of this region has not been sequenced. The partial sequenceof the U2B" genomic clone is shown in the sequence listing as threeseparate sequences since incomplete sequence data is available for thispromoter. The first of the U2B" sequences shown is SEQ:ID:No2 whichcorresponds to the region from the 5' end TCT to a TAA 119 basesdownstream. After the 3' end of SEQ:ID:No2 there is a region ofapproximately 900 bases which have not been sequenced. After the 3' endof the 900 bp unidentified region, the next U2B" sequence portionavailable is SEQ:ID:No3 which runs from a TCT approximately 900 basesdownstream of the end of SEQ:ID:No2 to an ATC a further 139 basesdownstream. After the 3' end of SEQ:ID:No3 there is a region of 11 baseswhich have not been identified. This unidentified region is followed bythe last sequenced portion of the U2B" clone which is designated asSEQ:ID:No4 which starts at ATA at its 5' end and ends at the ATG codon(bases 729-731) initiating transcription of the coding region of U2B"(not shown).

Spliceosomal protein gene promoters (or other expression controlpolynucleotides) have the advantages that (a) spliceosomal proteins areabsolutely required and thus spliceosomal protein gene promoters arelikely to be active in every cell and tissue type; (b) they are notderived from infectious agents which overcomes objections to the use ofsuch sequences due to potential recombination; and (c) different genesor DNA sequences are likely to be expressed at different levelsreflecting the relative abundance of the different spliceosomalproteins.

Modifications and improvements may incorporated without departing fromthe scope of the invention. For example, an expression controlpolynucleotide in accordance with the invention may be operably linkedto a second polynucleotide which has some function other than coding fora polypeptide. One such example might be to operably link an expressioncontrol polynucleotide to a gene encoding a ribozyme or anti-sense RNA.Indeed it will be realised by the skilled man that the function of thegene under the control of the expression control polynucleotide of theinvention is not important, and that the expression of many diversegenes can be controlled.

References

The following references are incorporated herein by reference.

Jackson, S. P., Lossky, M & Beggs, J. D. (1988). Mol Cell Biol. 8,1067-1075.

Jefferson, R. A. (1987). Assaying chimaeric genes in plants: the GUSgene fusion system. Plant Molecular Biology Reporter 5, 387-405.

Simpson, G. G., Vaux, P., Clark, G., Waugh, R., Beggs, J. D. & Brown, J.W. S. (1991). Evolutionary conservation of the spliceosomal protein,U2B". Nucleic Acids Research 19, 5213-5217.

Simpson, C. G., Sinibaldi, R. & Brown, J. W. S. (1992). Rapid analysisof plant gene expression by a novel reverse transcriptase-PCR method.Plant Journal 2, 835-836.

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning--ALaboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

Shaw, C. H. (1988). Plant molecular biology: a practical approach. IRLPress.

                  APPENDIX 1                                                      ______________________________________                                        TO-                TO+                                                                             4 ml solution 1  4 ml solution 1                           200 ml solution 3  4 ml solution 2                                            200 ml solution 4 200 ml solution 3                                           200 ml NAA (3 mg/ml) 200 ml solution 4                                        200 ml BAP (1 mg/ml) 200 ml NAA (3 mg/ml)                                      16 g Mannitol 200 ml BAP (1 mg/ml)                                           pH 5.5 adjusted with NaOH  16 g Mannitol                                      filtersterilised +200 ml  4 g Sucrose                                         cefotaxime (100 mg/ml)  40 ml Tween 20                                         pH 5.5 adjusted with NaOH                                                     filtersterilised +200 ml                                                      cefotaxime (100 mg/ml)                                                       Solution 1 Solution 2                                                         10.30 mM NH.sub.4 NO.sub.3 100 μM FeSO.sub.4                                9.40 mM KNO.sub.3 100 μM Na.sub.2 EDTA                                     1.50 mM CaCl.sub.2 2H.sub.2 O                                                 0.75 mM MgSO.sub.4.7H.sub.2 O                                                 0.62 mM KH.sub.2 PO.sub.4                                                    Solution 3 Solution 4                                                         16.00 μM H.sub.3 BO.sub.3 555.00 μM Inostol                              0.60 μM MnSO.sub.4.H.sub.2 O  3.00 μM Thiamine                          3.50 μM ZnSO.sub.4.7H.sub.2 O  5.00 μM Pyridoxine                       0.12 μM CuSO.sub.4.5H.sub.2 O  8.00 μM Niacin                           0.22 μM AlCl.sub.3  2.00 μM Pantothenate                                0.13 μM NiCl.sub.3.6H.sub.2 O  0.04 μM Biotine                          0.06 μM KI    +1 mg/μl NaOH                                          ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 784 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - TTAGAATTAG AATCCCCATT TTTAAGAATA ATCCTAGATA ATTTTCTTAA AC -            #ATGACAAT     60                                                                 - - TGATACCCAC AATTAATTAC TATTACATAA ATTTTTACCT AAATTAGATA TA -            #ACTTTCAA    120                                                                 - - TTTCAAAAAT TAAAACCCAA AAAAATTGAA CGACAATACG AGAGGGGATC AA -            #ACATAGGC    180                                                                 - - GAGCAATTAG AGAAATTGAC GGGTAGACAT CAACAAACCA TCAAGAATTT AA -            #AAGCGGAA    240                                                                 - - AGAGAAAAAA ATACACTATG GACGAATATT TTTATAGAAT TCAATATGTA AA -            #ACTAATAA    300                                                                 - - ACAAGAAAGT AAATCATCTT TTATTCAAAG TAATGAAGAA GAAGAATTGA AT -            #AAATATTT    360                                                                 - - ACATAATCAA TAAAAAAAAC TCATTCAAAA GAATCGTGTG TATGGGAAAG AA -            #GAAGAAAA    420                                                                 - - AAAAGGCAGA AAAAAACCAC TTCCCAATAA AAAAGGACAT CATGCTGCCA CC -            #TCCTAAAA    480                                                                 - - TTATTTAATT TAATTAAAAA AAAAACTTCC CAACACGTGG GCTACTAATT GC -            #AAAATTTA    540                                                                 - - ATTTTTAAAA AGCTTTTTTT GTCAAGAAAA TAAAAGATGG CTATATGTTG CC -            #AATTAGTA    600                                                                 - - AAATGGGATG TCATGCTGTG TCATTTTTTC TTGAGTTGTT AAGGGCTCAA AG -            #CCCAATTG    660                                                                 - - TTTATCCAGC CCAAGCCCAA ATCGGAGCCC TATTCGTGCC CAAAAATTTC TG -            #GAGAAATT    720                                                                 - - AACGACAACT GAAGTTTCTA CCTCACCGGC GAAAGTTGCA GCTAGGCGTA GA -            #CGAGGAGC    780                                                                 - - CATG                 - #                  - #                  - #                784                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - TCTCTCCAGC TCTTCCCTCC TAAAACAACC ATTTTATGAG TACAGACACA AA -             #CCAGCTTA     60                                                                 - - GCAACCAGTA AATCCAAAAC TTTAATTCCA CGTGTAAGCG CTAACACTTC AC -            #CCACTAA     119                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 139 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - TCTTTACACT ATTTTCAAAT AACGATGAGA CCTGTAATAA TGTAATAACT TG -            #AAAATAGA     60                                                                 - - ACAATAACTC ATTCAGTACA ACAAATAAAA TCATACTAAT GTATATTTTT AA -            #AAACAATT    120                                                                 - - TAACTCTATT TAATTAATC             - #                  - #                      - #139                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 731 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - ATATCTAAAA AAATGTGATT GGAGCACTCA TTGACCACAC GAATGAATCT AC -             #AATGTAGA     60                                                                 - - TCATTCTATC TAGACAAATA ATGCAAAAAC TAAAAGATAA AAGTAATATT AT -            #ACTACAAT    120                                                                 - - CTGTTAAATG ATATCAATAT TACAAAAGTT CTCTACGATG TCAATACATA TA -            #TTAAAATC    180                                                                 - - TATTTGATTA ATCAGAAACA TATCATGTGT GAATTTTTTT AATTAAAGAT CC -            #CTTTAATC    240                                                                 - - ATCTGAATCA ACCTTGGCTG GTCTCACATC TTTCCACCCT CTACTCGGTC TT -            #CACTTTCT    300                                                                 - - CTTAAACTAG GGAAGAACAA CATGATATTA GCTTAGATTA ATTAAACAAG CT -            #CATCAAAA    360                                                                 - - CTACCATCCA ATTTAAGCCA ATAATGTTTA AATAAAACAA AAAACAACGT AC -            #TCATTTTT    420                                                                 - - TCATAACAAG AAGTTAAAAT TTATATGAAT CCTTACTCCA AAAAAGAAGA AA -            #AATTACAA    480                                                                 - - TATCAATATA TATAACTACT CTATTTGGTT AGTCAACAAA ATGTTAGTAT AT -            #GTATTGCA    540                                                                 - - AGTTCGCAAC ACCCGCTTGG GCCTTGACCA CATATTTATA TGGGCCGGTT GT -            #CAATTTAA    600                                                                 - - GCCCACTTTG TGTTCGTTCG CCTTTCTTGT AGCTCCAAAC TCTTGGAAAT TT -            #GTCGAGCA    660                                                                 - - CATTCAGAAA TCACAGAGAA GAGCAAGTGA ATATACATAC AGATAGAGAA AA -            #GCTGCTCT    720                                                                 - - GCTCGGTAAT G               - #                  - #                      - #      731                                                                __________________________________________________________________________

We claim:
 1. An isolated polynucleotide comprising a spliceosomal protein gene promoter of a plasmid in a cell line deposited as any one of NCTC 12864, NCTC 12865, or NCTC
 12866. 2. A recombinant polynucleotide comprising the spliceosomal protein gene promoter according to claim
 1. 3. A recombinant vector comprising the spliceosomal protein gene promoter according to claim
 1. 4. A host cell transformed with the spliceosomal protein gene promoter as claimed in claim
 1. 5. A host cell comprising the recombinant polynucleotide according to claim
 2. 6. A host cell comprising the recombinant vector according to claim
 3. 7. An isolated polynucleotide comprising a spliceosomal protein gene promoter of a plasmid in NCTC 12864 between an ATG codon at a start of a coding sequence and a PstI site 2.5 kb upstream of the ATG codon.
 8. An isolated polynucleotide comprising a spliceosomal protein gene promoter of a plasmid in NCTC 12865 between an ATG codon at a start of a coding sequence and an EcoRI site 2 kb upstream of the ATG codon.
 9. A recombinant expression control polynucleotide comprising a promoter selected from the group of plant spliceosomal protein gene promoters consisting of potato U1A promoter, potato U2B"₋₋ promoter, and maize PRP8 promoter.
 10. A recombinant polynucleotide comprising the expression control polynucleotide according to claim 9 operably linked to a second polynucleotide.
 11. The recombinant polynucleotide according to claim 10, wherein the second polynucleotide encodes a polypeptide.
 12. The recombinant polynucleotide according to claim 10, wherein the second polynucleotide encodes a ribozyme.
 13. The recombinant polynucleotide according to claim 10, wherein the second polynucleotide encodes anti-sense RNA.
 14. A host cell comprising the recombinant polynucleotide according to claim
 10. 15. A transgenic plant or the progeny or seeds thereof, comprising the recombinant polynucleotide according to claim
 10. 16. A recombinant vector comprising the expression control polynucleotide according to claim
 9. 17. A method for producing a recombinant vector, said method comprising ligating the expression control polynucleotide according to claim 9 to a vector.
 18. A method for controlling the expression of a polypeptide from a polynucleotide encoding the polypeptide, said method comprising operably linking said polynucleotide to the expression control polynucleotide according to claim 9 and permitting expression of the polynucleotide encoding the polypeptide.
 19. A recombinant polynucleotide comprising a sequence selected from the group consisting of SEQ ID No:1,SEQ ID No:2, SEQ ID No:3, and SEQ ID No4.
 20. A recombinant polynucleotide comprising the polynucleotide according to claim 19, operably linked to a second polynucleotide.
 21. The recombinant polynucleotide according to claim 20, wherein the second polynucleotide encodes a polypeptide.
 22. The recombinant polynucleotide according to claim 20, wherein the second polynucleotide encodes a ribozyme.
 23. The recombinant polynucleotide according to claim 20, wherein the second polynucleotide encodes anti-sense RNA.
 24. A recombinant vector comprising at least one polynucleotide according to claim
 19. 25. A host cell comprising the recombinant polynucleotide according to claim
 20. 26. A transgenic plant or the progeny or seeds thereof, comprising the recombinant polynucleotide according to claim
 20. 27. A method for producing a recombinant vector, said method comprising ligating the expression control polynucleotide according to claim 19 to a vector.
 28. A method for controlling the expression of a polypeptide from a polynucleotide encoding the polypeptide, said method comprising operably linking said polynucleotide to the expression control polynucleotide according to claim 19 and permitting expression of the polypeptide encoded by said polynucleotide. 